Affinity chromatography matrix with built-in reaction indicator

ABSTRACT

p-Nitrophenylester of succinoylaminopropyl silica gel is useful as an affinity matrix for reacting with ligand for use in affinity chromatography. The affinity matrix, upon reaction with a ligand, releases one molecule of p-nitrophenolate ion for every molecule of ligand that reacts and the solution turns yellow as the reaction occurs.

FIELD OF THE INVENTION

This invention relates to the field of affinity chromatography and toaffinity matrixes for use in affinity chromatography.

BACKGROUND OF THE INVENTION

Affinity chromatography is a separation technique based on specific andreversible molecular interactions between two biologically activesubstances. However, the development of affinity chromatography has beenretarded to a great extent by the absence of suitable supports and thelack of satisfactory techniques for immobilizing ligands.

It is especially desirable that an affinity matrix be available for usein affinity chromatography which maxtrix has a leaving group, used as anactivator for the coupling reaction with the ligand, which forms areaction indicator in aqueous solutions.

BRIEF SUMMARY OF THE INVENTION

It has been found that the p-nitrophenylester of succinoylaminopropylsilica gel as an affinity matrix is particularly useful for reactingwith ligands to produce an affinity matrix having a ligand covalentlybound to the affinity matrix and useful in affinity chromatography toseparate or purify a substance from a solution by reacting saidsubstance in the solution with the affinity matrix having a ligandcovalently bound to the affinity matrix. Additionally, it has been foundthat the use of the p-nitrophenylester of succinoylaminopropyl silicagel as an affinity matrix provides a matrix having a leaving group usedas an activator for the coupling reaction with a ligand which leavinggroup forms a bright yellow anion in aqueous solution at pH>7. Thus,this affinity matrix is highly beneficial in that it contains anactivated reactive group with a built-in or internal reaction indicator.

DETAILS OF THE INVENTION

The novel affinity matrix of this invention is the p-nitrophenylester ofsuccinoylaminopropyl silica gel of the general formula ##STR1## wherein##STR2## represents the silica gel. The p-nitrophenylester ofsuccinoylaminopropyl silica gel is prepared by reacting p-nitrophenolwith succinoylaminopropyl silica gel in the presence ofdicylohexylcarbodimide.

The succinoylaminopropyl silica gel is commercially available or can beprepared, for example, from silica gel by reacting silica gel withaminopropyltriethoxysilane and reacting the product aminopropyl silicagel with succinic anhydride. Silica gel suitable for use in forming thematrix of this invention is any particulate silica gel having an averageparticle diameter of from about 3 to about 200 microns and an averagepore size of from about 50 to about 1000 Angstrom units. Such silicagel, consisting of amorphous silica, is commercially available inirregular and spherical particulate forms. Where the affinity matrixproduct of this invention is to be used in chromatographic columns asilica gel of from about 3 to about 70 microns average diameter isgenerally employed whereas if the affinity matrix product of thisinvention is to be used in a batch separation medium, such as a testtube or the like, silica gel of from about 3 to about 200 micronsaverage diameter can be employed.

The p-nitrophenylester of succinoxylaminopropyl silica gel affinitymatrix of this invention can be used as an affinity matrix for bindingto any ligand which covalently bonds to the affinity matrix. Theaffinity matrix is especially useful for reacting with ligands havingreactive amino groups although it is also quite useful for reacting withligands having other reactive groups such as for example ligandscontaining reactive hydroxyl, sulfhydryl and the like groups. Thenitrophenyl ester of the matrix of this invention readily reacts withsuch a reactive groups of a protein, enzyme or other such ligand toyield the ligand immobilized on the silica gel matrix. As examples ofligands containing such reactive groups which can be immobilized on theaffinity matrix of this invention by covalent binding thereto there canbe mentioned for example antigens, antibodies, enzymes, inhibitors,cofactors, hormones, vitamins, toxins, growth factors, glycoconjugates,lectins, nucleic acids and proteins which are known in the art. Theligand bound affinity matrixes of this invention are employed to purifyor separate substances, such as for example, proteins containingreactive amine groups, from solutions containing such substance byreacting the substance in solution with an affinity matrix of thisinvention having a ligand covalently bound to the affinity matrix. Amongsuch substances to be separated or purified there can be mentioned, forexample, trypsin, d-tryptophan methyl ester, m-aminobenzamidine,p-aminobenzyl-thio-N-acetyl-β-d-glucosamine, p-aminophenyllactic acid,phenylbutylamine and the like.

The p-nitrophenylester of succinoylaminopropyl silica gel affinitymatrix of this invention has the special advantage that the leavinggroup used as the activator for the coupling reaction with the ligandforms a bright yellow anion in aqueous solutions at pH>7. Therefore, asthe ligand-matrix coupling reaction occurs, for every molecule of ligandthat reacts, one molecule of p-nitrophenolate ion, absorptionε˜10,000 at400 nm wavelength, is released and the solution turns yellow as thereaction occurs. At neutral pH's the p-nitrophenylester ofsuccinoylaminopropyl silica gel is only very slowly reactive with water,and is very reactive with amine groups and other nucleophiles.Therefore, formation of yellow color is an accurate measure of the rateand extent of ligand reaction.

The affinity matrix of this invention and the use thereof to covalentlybond to ligands is illustrated but not limited by the followingexamples.

Although succinoylaminopropyl silica gel is commercially available andcould be used for the preparation of the affinity matrix of thisinvention, it is possible that the succinoylaminopropyl silica gelreactant can be prepared according to the following exemplarypreparation.

PREPARATION

Fifty grams of silica gel (40 u, 650 A pore), was reacted with 22.5grams of aminopropyltriethoxysilane in about 250 ml of toluene for about20 hours. After filtration, the solid silica gel was washed with 200 mlof toluene twice, 200 ml of methanol twice and 200 ml of ethyl etheronce. It was dried and heat treated at about 80° C. for about 4 hours,43 minutes.

The aminopropyl silica gel product was mixed with 300 ml of toluene and16 grams of succinic anhydride and rotated in a 40° C. bath for about21/2hours, filtered, washed with 200 ml of toluene twice, 200 ml ofmethanol twice and 200 ml of ethyl ether once and oven dried.

Treatment of the succinoylaminopropyl silica gel produced with 15 ml oftrimethylchlorosilane in 300 ml of toluene followed by filtration,washing and oven drying yielded a capped succinoylaminopropyl silica gelproduct with a titer of 0.3 meq of acid groups per gram.

EXAMPLE 1

15 grams of the succinoylaminopropyl silica gel product of the abovePreparation was treated in about 114 ml of toluene and 7.5 ml of drypyridine with 0.95 grams p-nitrophenol and 3.8 grams of dicyclohexylcarbodiimide and 0.2 g dimethylamino pyridine for about 2 hours, 18minutes. The reaction mixture was filtered, washed with 100 ml oftoluene twice, and with 100 ml of methanol twice, and then dried in anoven at 80° C. About 15.3 grams of the p-nitrophenylester ofsuccinoylaminopropyl silica gel product resulted.

EXAMPLE 2

The p-nitrophenylester of succinoylaminopropyl silica gel affinitymatrix of Example 1 was treated with 200 mg of trypsin dissolved in 60ml of phosphate buffer (pH 7) on a shaker for about 4 hours and thenabout 62 ml of 0.1 M tris (trihydroxymethyl) aminomethane solution (pH8.0) for about 3 hours. After filtration and washing with pH 7 buffer,it was stored in pH 7 buffer. Tested for activity, the product showedhigh trypsin activity.

EXAMPLE 3

In this example, the reaction of trypsin with the p-nitrophenylester ofsuccinoylaminopropyl silica gel affinity matrix of this invention ismonitored by following the absorbance of p-nitrophenol in the reactionsupernatant. Concomitantly the extent of the reaction is measured bymeasuring the amount of trypsin remaining in the supernatant bymeasuring the absorbance of the solution at 2 nm wavelength. When theabove two absorbance measurements were made, the absorbance at 280 nm ofthe trypsin in the supernatent, as compared with a suitable reagentblank, decreased rapidly to zero over the same time period as theabsorbance of p-nitrophenol, measured at 400 nm increased to a maximum.Correlation of the two values which is shown by this exampledemonstrates the validity and usefulness of employing the release ofp-nitrophenolate ion and the coloration of the aqueous solution therebyas an accurate measure of the rate and extent of ligand reaction withaffinity matrix.

We claim:
 1. The p-nitrophenylester of succinoylaminopropyl silica gel.2. The product of claim 1 wherein the silica gel has an average particlediameter of from about 3 to about 70 microns and an average pore size offrom about 50 to about 100 Angstrom units.
 3. In a method of separatingor purifying a substance from a solution by reacting the substance inthe solution with an affinity matrix having a ligand covalently bound tothe affinity matrix, the improvement comprising employing thep-nitrophenylester of succinoylaminopropyl silica gel having a ligandcovalently bound thereto as the affinity matrix.
 4. The method of claim3 wherein the silica gel affinity matrix has an average particlediameter of from about 3 to about 70 microns.
 5. The method of claim 3wherein the ligand is a protein containing an amine group reactive withthe affinity matrix.
 6. The method of claim 4 wherein the ligand is aprotein containing an amine group reactive with the affinity matrix.